Ciona Intestinalis Research Articles

Sperm viability assessment in marine invertebrates by fluorescent staining and spectrofluorimetry: A promising tool for assessing marine pollution impact

The viability of spermatozoa is a crucial parameter to evaluate their quality that is an important issue in ecotoxicological studies. Here, a new method has been developed to rapidly determine the viability of spermatozoa in three marine invertebrates: the ascidian Ciona intestinalis, the sea urchin Paracentrotus lividus and the mollusc Mytilus galloprovincialis. This method employed the dual DNA fluorescent staining coupled with spectrofluorimetric analysis. The dual fluorescent staining used the SYBR-14 stained live spermatozoa and propidium iodide stained degenerated cells that had lost membrane integrity. Stain uptake was assessed by confocal microscopy and then the percentage of live and dead spermatozoa was quantified by spectrofluorimetric analysis. The microscopic examination revealed three populations of spermatozoa: living-SYBR-14 stained, dead-PI stained, and dying-doubly stained spermatozoa. The fluorescence emission peak values recorded in a spectrofluorimeter provide the portion of live and dead spermatozoa showing a significant negative correlation. The stain combination was further validated using known ratios of live and dead spermatozoa.The present study demonstrated that the dual DNA staining with SYBR-14 and propidium iodide was effective in assessing viability of spermatozoa in marine invertebrates and that spectrofluorimetric analysis can be successfully employed to evaluate the percentage of live and dead spermatozoa. The method develop herein is simple, accurate, rapid, sensitive, and cost-effective, so it could be a useful tool by which marine pollutants may be screened for spermiotoxicity.

Invertebrate Gonadotropin-Releasing Hormone-Related Peptides and Their Receptors: An Update.

Gonadotropin-releasing hormones (GnRHs) play pivotal roles in reproductive functions via the hypothalamus, pituitary, and gonad axis, namely, HPG axis in vertebrates. GnRHs and their receptors (GnRHRs) are likely to be conserved in invertebrate deuterostomes and lophotrochozoans. All vertebrate and urochordate GnRHs are composed of 10 amino acids, whereas protostome, echinoderm, and amphioxus GnRH-like peptides are 11- or 12-residue peptide containing two amino acids after an N-terminal pyro-Glu. In urochordates, Halocynthia roretzi GnRH gene encodes two GnRH peptide sequences, whereas two GnRH genes encode three different GnRH peptides in Ciona intestinalis. These findings indicate the species-specific diversification of GnRHs. Intriguingly, the major signaling pathway for GnRHRs is intracellular Ca2+ mobilization in chordates, echinoderms, and protostomes, whereas Ciona GnRHRs (Ci-GnRHRs) are endowed with multiple GnRHergic cAMP production pathways in a ligand-selective manner. Moreover, the ligand-specific modulation of signal transduction via heterodimerization among Ci-GnRHR paralogs suggests the species-specific development of fine-tuning of gonadal functions in ascidians. Echinoderm GnRH-like peptides show high sequence differences compared to those of protostome counterparts, leading to the difficulty in classification of peptides and receptors. These findings also show both the diversity and conservation of GnRH signaling systems in invertebrates. The lack of the HPG axis in invertebrates indicates that biological functions of GnRHs are not release of gonadotropins in current invertebrates and common ancestors of vertebrates and invertebrates. To date, authentic or putative GnRHRs have been characterized from various echinoderms and protostomes as well as chordates and the mRNAs have been found to be distributed not only reproductive organs but also other tissues. Collectively, these findings further support the notion that invertebrate GnRHs have biological roles other than the regulation of reproductive functions. Moreover, recent molecular phylogenetic analysis suggests that adipokinetic hormone (AKH), corazonin (CRZ), and AKH/CRZ-related peptide (ACP) belong to the GnRH superfamily but has led to the different classifications of these peptides and receptors using different datasets including the number of sequences and structural domains. In this review, we provide current knowledge of, and perspectives in, molecular basis and evolutionary aspects of the GnRH, AKH, CRZ, and ACP.

Baseline and oxidative DNA damage in marine invertebrates

ABSTRACTAnthropogenic pollutants produce oxidative stress in marine organisms, directly or following generation of reactive oxygen species (ROS), potentially resulting in increased accumulation of DNA strand breaks quantified. The aim of this study is to quantify baseline levels of DNA strand breaks in marine species from four phyla and to assess relative sensitivity to oxidative stress as well as ability to recover. DNA strand breaks were determined using a formamidopyrimidine DNA glycosylase (Fpg)-amended comet assay in circulating cells from blue mussel (Mytilus edulis), shore crab (Carcinus maenas), sea star (Asterias rubens), and vase tunicate (Ciona intestinalis). Lymphocytes from Atlantic cod (Gadus morhua) were used as a reference. In addition to immediate analysis, cells from all species were exposed ex vivo to two concentrations of hydrogen peroxide (H2O2) at 25 or 250 μM prior to assay. Mean baseline DNA strand breaks were highest for cells from sea star (34%) followed by crab (25%), mussel (22%), tunicate (17%), and cod (14%). Circulating cells from invertebrates were markedly more sensitive to oxidative stress compared to cod lymphocytes. DNA strand breaks exceeded 80% for sea star, crab, and mussel cells following exposure to the lowest H2O2 concentration. There was no recovery for cells from any species following 1 hr in buffer. This study provides an in-depth analysis of DNA integrity for ecologically important species representing 4 phyla. Data indicate that circulating cells from invertebrates are more sensitive to oxidative stress than cells from fish as evidenced by DNA strand breaks. Future studies need to address the extent to which DNA strand breaks may exert consequences for body maintenance costs in marine invertebrates.

33 ホヤ精子の活性化・誘引物質の化学合成と微量構造決定(口頭発表の部)

33 ホヤ精子の活性化・誘引物質の化学合成と微量構造決定(口頭発表の部)

Self-fertilization and inbreeding depression in three ascidian species that differ in genetic dispersal potential

Although self-fertilization can mitigate the costs of sexual reproduction, many hermaphroditic marine invertebrates avoid selfing, presumably because of inbreeding depression. Eventually, successive generations of inbreeding are expected to reduce genetic load, but selfing cannot evolve for future benefits. Initial inbreeding costs of selfing could be offset by the immediate benefits of local adaptation and mating assurance, both of which are more likely in species with limited gene flow. We compared the likelihood of selfing and the magnitude of inbreeding depression among three ascidian species (Molgula provisionalis, Ciona intestinalis, and Botryllus schlosseri) that were known a priori to differ in larval dispersal potential. Selfing potential exhibited a negative association, and inbreeding depression a positive association with dispersal potential. M. provisionalis, with highly philopatric larvae, had no apparent barrier to self-fertilization and exhibited little evidence of inbreeding depression (ratio of survival of self to outcross progeny 10 weeks after metamorphosis was 0.999). By contrast, C. intestinalis, with highly dispersive larvae, exhibited low levels of self-fertilization in flowing water and high levels of inbreeding depression (survival ratio 14 weeks after metamorphosis of 0.274). B. schlosseri larvae have intermediate dispersal capabilities, yet exhibited low likelihood of self-fertilization and high magnitude of inbreeding depression (survival ratio 10 weeks after metamorphosis of 0.310); however, extremely long-lived sperm contribute to gene flow in B. schlosseri. These results suggest that in marine hermaphrodites, gene flow, self-fertilization, and inbreeding depression should be evaluated as an integrated suite of traits, not independent characters.

Establishment of lateral organ asymmetries in the invertebrate chordate, Ciona intestinalis

BackgroundThe evolutionary emergence and diversification of the chordates appear to involve dramatic changes in organ morphogenesis along the left/right axis. However, the ancestral chordate mechanism for establishing lateral asymmetry remains ambiguous. Additionally, links between the initial establishment of lateral asymmetry and subsequent asymmetries in organ morphogenesis are poorly characterized.ResultsTo explore asymmetric organ morphogenesis during chordate evolution, we have begun to characterize left/right patterning of the heart and endodermal organs in an invertebrate chordate, Ciona intestinalis. Here, we show that Ciona has a laterally asymmetric, right-sided heart. Our data indicate that cardiac lateral asymmetry requires H+/K+ ion flux, but is independent of Nodal signaling. Our pharmacological inhibitor studies show that ion flux is required for polarization of epidermal cilia and neurula rotation and suggest that ion flux functions synergistically with chorion contact to drive cardiac laterality. Live imaging analysis revealed that larval heart progenitor cells undergo a lateral shift without displaying any migratory behaviors. Furthermore, we find that this passive shift corresponds with the emergence of lateral asymmetry in the endoderm, which is also ion flux dependent.ConclusionsOur data suggest that ion flux promotes laterally asymmetric morphogenesis of the larval endoderm rudiment leading to a passive, Nodal-independent shift in the position of associated heart progenitor cells. These findings help to refine hypotheses regarding ancestral chordate left/right patterning mechanisms and how they have diverged within invertebrate and vertebrate chordate lineages.

Conserved gene regulatory module specifies lateral neural borders across bilaterians

The lateral neural plate border (NPB), the neural part of the vertebrate neural border, is composed of central nervous system (CNS) progenitors and peripheral nervous system (PNS) progenitors. In invertebrates, PNS progenitors are also juxtaposed to the lateral boundary of the CNS. Whether there are conserved molecular mechanisms determining vertebrate and invertebrate lateral neural borders remains unclear. Using single-cell-resolution gene-expression profiling and genetic analysis, we present evidence that orthologs of the NPB specification module specify the invertebrate lateral neural border, which is composed of CNS and PNS progenitors. First, like in vertebrates, the conserved neuroectoderm lateral border specifier Msx/vab-15 specifies lateral neuroblasts in Caenorhabditis elegans Second, orthologs of the vertebrate NPB specification module (Msx/vab-15, Pax3/7/pax-3, and Zic/ref-2) are significantly enriched in worm lateral neuroblasts. In addition, like in other bilaterians, the expression domain of Msx/vab-15 is more lateral than those of Pax3/7/pax-3 and Zic/ref-2 in C. elegans Third, we show that Msx/vab-15 regulates the development of mechanosensory neurons derived from lateral neural progenitors in multiple invertebrate species, including C. elegans, Drosophila melanogaster, and Ciona intestinalis We also identify a novel lateral neural border specifier, ZNF703/tlp-1, which functions synergistically with Msx/vab-15 in both C. elegans and Xenopus laevis These data suggest a common origin of the molecular mechanism specifying lateral neural borders across bilaterians.

Cultivating Ciona intestinalis to counteract marine eutrophication: Environmental assessment of a marine biomass based bioenergy and biofertilizer production system

Abstract Eutrophication in the North and Baltic Seas is a major problem to the marine environment and the communities depending on it. To counteract this, the Swedish Marine and Water Authority suggested financial support for measures that increase the uptake of nutrients from the water by e.g. marine organisms and support for the utilisation of these organisms as value added products. In Sweden the use of biogas to replace fossil transportation fuels is widely adopted. The domestic biogas production corresponded to approx. 1.95 TWh (approx. 7010 TJ) in 2015 of which approx. 63% were upgraded for use as e.g. transportation fuel. Other uses are heat and electricity generation as well as industrial applications. To expand production, the biogas industry is searching for new substrates. In this paper the utilisation of the marine evertebrate organism Ciona intestinalis (tunicata), cultivated in the North Sea and used as feedstock for biogas and biofertilizer production is suggested and assessed. The greenhouse gas (GHG) emissions performance of the concept and it's consequences on marine eutrophication are investigated applying life cycle assessment. Results show that at full scale biogas production from C. intestinalis reduces GHG emissions by more than 65% compared to fossil transportation fuels. In addition, the results show that accounting for the system consequences of other products and services such as biofertilizer replacing mineral fertilizers and decreased marine eutrophication largely increase the environmental benefits provided by the concept. Approx. 3.7 g-Neq/MJbiogas of nitrogen are removed from the marine environment during the cultivation of C. intestinalis.

Expression of Ciona intestinalis AOX causes male reproductive defects in Drosophila melanogaster

BackgroundMitochondrial alternative respiratory-chain enzymes are phylogenetically widespread, and buffer stresses affecting oxidative phosphorylation in species that possess them. However, they have been lost in the evolutionary lineages leading to vertebrates and arthropods, raising the question as to what survival or reproductive disadvantages they confer. Recent interest in using them in therapy lends a biomedical dimension to this question.MethodsHere, we examined the impact of the expression of Ciona intestinalis alternative oxidase, AOX, on the reproductive success of Drosophila melanogaster males. Sperm-competition assays were performed between flies carrying three copies of a ubiquitously expressed AOX construct, driven by the α-tubulin promoter, and wild-type males of the same genetic background.ResultsIn sperm-competition assays, AOX conferred a substantial disadvantage, associated with decreased production of mature sperm. Sperm differentiation appeared to proceed until the last stages, but was spatially deranged, with spermatozoids retained in the testis instead of being released to the seminal vesicle. High AOX expression was detected in the outermost cell-layer of the testis sheath, which we hypothesize may disrupt a signal required for sperm maturation.ConclusionsAOX expression in Drosophila thus has effects that are deleterious to male reproductive function. Our results imply that AOX therapy must be developed with caution.

Консорты брюхоногого моллюска Rapana venosa (Valenciennes, 1846) в северной части Чёрного моря. Часть I: Porifera, Cnidaria, Bryozoa, Chordata

Брюхоногий моллюск-вселенец Rapana venosa (Valenciennes, 1846) в 1940-х занял свободную экологическую нишу терминального хищника в бентосных сообществах шельфа Чёрного моря и стал важным элементом его экосистемы. Способность R. venosa оказывать разрушительное воздействие на биоценозы фильтраторов определяет необходимость постоянного мониторинга состояния популяций этого вида. Однако его экологическая роль не сводится только к хищничеству. Довольно крупная раковина R. venosa служит твёрдым субстратом для водорослей, прикреплённых и малоподвижных (седентарных) бентосных животных организмов, среди которых иногда находят убежище и подвижные свободноживущие формы. Цель работы — описание специфического комплекса эпибионтных организмов на раковине рапаны, который рассматривается как консорция, где R. venosa является ядром. Для изучения консортного сообщества R. venosa были осуществлены сборы рапаны в 7 районах северной части Чёрного моря: 1 — пляж Мамайя, Румыния; 2 — северо-западная часть Чёрного моря (СЗЧМ), Крымский сектор; 3 — Севастополь; 4 — Алупка; 5 — Ялта — Алушта; 6 — Карадаг; 7 — Керченский пролив. Сбор материала в прибрежной зоне (до 15 м) проводился легководолазным способом, на больших глубинах (до 40 м) использовался дночерпатель «Океан-50». Всего было собрано и проанализировано 856 экземпляров R. venosa. Основной объём материала в количестве 750 экз. R. venosa собран в прибрежной зоне г. Севастополя (Юго-Западный Крым) в диапазоне глубин 1.5–10.0 м. Сбор особей рапаны проводили тотально, каждый экземпляр помещали в отдельный пластиковый пакет с указанием района сбора, глубины и биотопа. Параллельно со сбором моллюсков проводили визуальные наблюдения и фотофиксацию гидробионтов in situ. По результатам исследований расширен таксономический список эпибионтных организмов рапаны, впервые приводится таксономический состав подвижных форм, постоянно или временно присутствующих в консорции R. venosa. Представленная первая часть работы посвящена эпибионтам рапаны, относящимся к четырём (Porifera, Cnidaria, Bryozoa, Chordata) из семи обнаруженных типов животных, с указанием глубин обнаружения и типа грунта, а также комментариями по встречаемости, количеству обнаруженных видов и их взаимодействию с ядром консорции. Тип Porifera представлен 1 видом, Cnidaria — 2, Bryozoa — 5, Chordata — 5 видами. Сверлящая губка Pione vastifica обнаружена во всех исследованных районах кроме р-на 2 (СЗЧМ) со встречаемостью от 20 до 90 % и площадью повреждения раковин до 100 % (в среднем 30–35 %). Представители Cnidaria обнаружены единично в р-не 3 (Actinia equina) и в р-не 5 (Diadumene lineata). Мшанки — наиболее распространённая группа животных — консортов рапаны, присутствующая во всех районах исследований. Их встречаемость варьировала от 10 до 100 %, площадь покрытия раковины — от 0 до 85 %. Более 90 % в общей площади покрытия раковин рапаны мшанками и их встречаемости приходится на Cryptosula pallasiana. Вторым по показателям развития видом мшанок является Conopeum seurati, который обнаружен в р-нах 3, 6 и 7, где имел встречаемость до 30 % и площадь покрытия до 25 %. Остальные 3 вида мшанок (Schizomavella auriculata, Bowerbankia imbricata, Hippothoa sp.) встречались редко. Представители Chordata — 2 вида асцидий (Botryllus schlosseri, Ciona intestinalis) и 3 вида рыб (Diplecogaster bimaculata, Parablennius tentacularis, Scorpaena porcus) — зафиксированы единично в р-нах 2, 3, 5. Для рассмотренных четырёх типов животных организмов список консортов R. venosa в биотопе рыхлых грунтов (10 видов) в два раза больше, чем у скальной экоформы (5 видов). Рассмотренные организмы имеют различную степень и характер связей с ядром консорции — от комменсализма до паразитизма. Дана обобщённая информация о водорослевых обрастаниях и суммарном покрытии раковин R. venosa комплексом эпибионтов. Результаты работы показывают, что R. venosa в Чёрном море стала значимым элементом экосистемы, увеличивающим биологическое разнообразие донной фауны за счёт формирования собственных консортивных комплексов эпибионтных организмов.

Shared hemocyte- and intestine-dominant expression profiles of intelectin genes in ascidian Ciona intestinalis: insight into the evolution of the innate immune system in chordates.

Intelectin is a soluble lectin known as a pattern-recognition receptor for the innate immune system or as an intestinal lactoferrin receptor. Intelectin genes have been identified in a wide range of chordates and the shared expression pattern in their absorptive intestinal regions has been widely recognized. The chordate intelectins have a shared domain structure with a fibrinogen-related domain and an intelectin domain and an additional sequence has been reported only in ascidian Ciona intestinalis intelectins. However, little is known about the molecular features of the ascidian intelectins, including the distribution of the additional sequence in ascidians. Therefore, we focus on the ascidian species that are available for genome DNA sequence searches and survey intelectin genes with special reference to the additional sequence. We also assess the distribution of Ciona intelectin gene transcripts in transparent juveniles and adult specimens by means of in situ hybridization and reveal hemocyte-dominant expressions as well as stomach-exclusive expression. Comparative gene expression analysis with secretory digestive enzymes and absorption-related proteins in Ciona revealed that intelectin and secretory digestive enzymes were expressed in the same region of the stomach epithelium. Since the domain structure of intelectins and the hemocyte-dominant gene expression of intelectins seem relevant to ficolin, intelectin genes may have evolved from a ficolin-like ancestral gene with hemocytic expression in early chordate evolution.

The nervous system of the adult ascidian Ciona intestinalis Type A (Ciona robusta): Insights from transgenic animal models.

The nervous system of ascidians is an excellent model system to provide insights into the evolutionary process of the chordate nervous system due to their phylogenetic positions as the sister group of vertebrates. However, the entire nervous system of adult ascidians has yet to be functionally and anatomically investigated. In this study, we have revealed the whole dorsal and siphon nervous system of the transgenic adult ascidian of Ciona intestinalis Type A (Ciona robusta) in which a Kaede reporter gene is expressed in a pan-neuronal fashion. The fluorescent signal of Kaede revealed the innervation patterns and distribution of neurons in the nervous system of Ciona. Precise microscopic observation demonstrated the clear innervation of the anterior and posterior main nerves to eight and six lobes of the oral and atrial siphons, respectively. Moreover, visceral nerves, previously identified as unpaired nerves, were found to be paired; one nerve was derived from the posterior end of the cerebral ganglion and the other from the right posterior nerve. This study further revealed the full trajectory of the dorsal strand plexus and paired visceral nerves on either side from the cerebral ganglion to the ovary, and precise innervation between the cerebral ganglion and the peripheral organs including the gonoduct, cupular organ, rectum and ovary. The differential innervation patterns of visceral nerves and the dorsal strand plexus indicate that the peripheral organs including the ovary undergo various neural regulations. Collectively, the present anatomical analysis revealed the major innervation of the dorsal and siphon nervous systems of adult Ciona.

Immunity in Protochordates: The Tunicate Perspective.

Tunicates are the closest relatives of vertebrates, and their peculiar phylogenetic position explains the increasing interest toward tunicate immunobiology. They are filter-feeding organisms, and this greatly influences their defense strategies. The majority of the studies on tunicate immunity were carried out in ascidians. The tunic acts as a first barrier against pathogens and parasites. In addition, the oral siphon and the pharynx represent two major, highly vascularized, immune organs, where circulating hemocytes can sense non-self material and trigger immune responses that, usually, lead to inflammation and phagocytosis. Inflammation involves the recruitment of circulating cytotoxic, phenoloxidase (PO)-containing cells in the infected area, where they degranulate as a consequence of non-self recognition and release cytokines, complement factors, and the enzyme PO. The latter, acting on polyphenol substrata, produces cytotoxic quinones, which polymerize to melanin, and reactive oxygen species, which induce oxidative stress. Both the alternative and the lectin pathways of complement activation converge to activate C3: C3a and C3b are involved in the recruitment of hemocytes and in the opsonization of foreign materials, respectively. The interaction of circulating professional phagocytes with potentially pathogenic foreign material can be direct or mediated by opsonins, either complement dependent or complement independent. Together with cytotoxic cells, phagocytes are active in the encapsulation of large materials. Cells involved in immune responses, collectively called immunocytes, represent a large fraction of hemocytes, and the presence of a cross talk between cytotoxic cells and phagocytes, mediated by secreted humoral factors, was reported. Lectins play a pivotal role as pattern-recognition receptors and opsonizing agents. In addition, variable region-containing chitin-binding proteins, identified in the solitary ascidian Ciona intestinalis, control the settlement and colonization of bacteria in the gut.

Developmental shift of Rx expression from non-photoreceptor to photoreceptor lineage as a mechanism for photoreceptor cell specification in Ciona intestinalis

Developmental shift of Rx expression from non-photoreceptor to photoreceptor lineage as a mechanism for photoreceptor cell specification in Ciona intestinalis

Compartmentalized expression patterns of pancreatic- and gastric-related genes in the alimentary canal of the ascidian Ciona intestinalis: evolutionary insights into the functional regionality of the gastrointestinal tract in Olfactores.

Many heterotrophic animals have a one-way alimentary canal that is essential for their nutrition and sequential steps of the digestive system, namely ingestion, digestion, absorption and elimination, are widely shared among bilaterians. Morphological, functional and molecular knowledge of the alimentary canal has been obtained in particular from mammalian research but the shared features and evolution of these aspects of the highly diverged alimentary canal in the animal kingdom are still unclear. We therefore investigate spatial gene expression patterns of pancreatic- and gastric-related molecules of ascidians (a sister group of vertebrates) with special reference to the functional regionality of the gastrointestinal tract. Genome-wide surveys of ascidian homologs to mammalian exocrine digestive enzyme genes revealed that pancreatic enzymes, namely alpha-amylase, lipase, phospholipase A2, trypsin, chymotrypsin and carboxypeptidase, exist in the ascidian genome. However, an ascidian homolog of the mammalian gastric enzyme pepsin has not been identified, although molecules resembling cathepsin D, a pepsin relative, are indeed present. Spatial expression analyses in the ascidian Ciona intestinalis, by means of whole-mount in situ hybridization, have elucidated that the expression of Ciona homologs of pancreatic- and gastric-related exocrine enzyme genes and of their transcriptional regulator genes is restricted to the Ciona stomach. Furthermore, the expression of these genes is localized to specific regions of the stomach epithelium according to their regionality in the vertebrate digestive system. The compartmentalized expression patterns of Ciona homologs imply primitive and/or ancestral aspects of molecular, functional and morphological bases among Olfactores.

Evolutionary steps involving counterion displacement in a tunicate opsin

Ci-opsin1 is a visible light-sensitive opsin present in the larval ocellus of an ascidian, Ciona intestinalis This invertebrate opsin belongs to the vertebrate visual and nonvisual opsin groups in the opsin phylogenetic tree. Ci-opsin1 contains candidate counterions (glutamic acid residues) at positions 113 and 181; the former is a newly acquired position in the vertebrate visual opsin lineage, whereas the latter is an ancestral position widely conserved among invertebrate opsins. Here, we show that Glu113 and Glu181 in Ci-opsin1 act synergistically as counterions, which imparts molecular properties to Ci-opsin1 intermediate between those of vertebrate- and invertebrate-type opsins. Synergy between the counterions in Ci-opsin1 was demonstrated by E113Q and E181Q mutants that exhibit a pH-dependent spectral shift, whereas only the E113Q mutation in vertebrate rhodopsin yields this spectral shift. On absorbing light, Ci-opsin1 forms an equilibrium between two intermediates with protonated and deprotonated Schiff bases, namely the MI-like and MII-like intermediates, respectively. Adding G protein caused the equilibrium to shift toward the MI-like intermediate, indicating that Ci-opsin1 has a protonated Schiff base in its active state, like invertebrate-type opsins. Ci-opsin1's G protein activation efficiency is between the efficiencies of vertebrate- and invertebrate-type opsins. Interestingly, the E113Y and E181S mutations change the molecular properties of Ci-opsin1 into those resembling invertebrate-type or bistable opsins and vertebrate ancient/vertebrate ancient-long or monostable opsins, respectively. These results strongly suggest that acquisition of counterion Glu113 changed the molecular properties of visual opsin in a vertebrate/tunicate common ancestor as a crucial step in the evolution of vertebrate visual opsins.

Differential gene expression along the animal-vegetal axis in the ascidian embryo is maintained by a dual functional protein Foxd.

In many animal embryos, a specific gene expression pattern is established along the animal-vegetal axis soon after zygotic transcription begins. In the embryo of the ascidian Ciona intestinalis, soon after the division that separates animal and vegetal hemispheres into distinct blastomeres, maternal Gata.a and β-catenin activate specific genes in the animal and vegetal blastomeres, respectively. On the basis of these initial distinct gene expression patterns, gene regulatory networks promote animal cells to become ectodermal tissues and vegetal cells to become endomesodermal tissues and a part of the nerve cord. In the vegetal hemisphere, β-catenin directly activates Foxd, an essential transcription factor gene for specifying endomesodermal fates. In the present study, we found that Foxd also represses the expression of genes that are activated specifically in the animal hemisphere, including Dmrt1, Prdm1-r.a (Bz1), Prdm1-r.b (Bz2), and Otx. A reporter assay showed that Dmrt1 expression was directly repressed by Foxd, and a chromatin immunoprecipitation assay showed that Foxd was bound to the upstream regions of Dmrt1, Prdm1-r.a, Prdm1-r.b, and Otx. Thus, Foxd has a dual function of activating specific gene expression in the vegetal hemisphere and of repressing the expression of genes that are normally expressed in the animal hemisphere. This dual function stabilizes the initial patterning along the animal-vegetal axis by β-catenin and Gata.a.

Transcriptomes of the Premature and Mature Ovaries of an Ascidian, Ciona intestinalis.

Transcriptomes of the Premature and Mature Ovaries of an Ascidian, Ciona intestinalis.

The invasive ascidian Ciona robusta recorded from a Red Sea marina

The solitary ascidian Ciona intestinalis is among the most damaging of invasive fouling species in the world. It is mostly known from cold-water or temperate environments, where it is able to form dense aggregations. We present the first record of C. intestinalis type-A, now recognized as C. robusta, from the Eilat marina, Israeli Red Sea coast. Several individuals found fouling the bottom of the floating docks in March 2015 were identified using both mitochondrial sequencing and morphological tools. Periodical surveys revealed some of them with full gonoducts, and small individuals appeared two months later, indicating a reproductive population. The extremely high salinity and temperature at the Eilat marina indicates the wide environmental tolerance of this species. Since the only other report from this region dates to 100 years ago, from a dredging vessel at the entrance to the Suez Canal, and it has not since been recorded from any natural substrates in the region, it should be considered a non-indigenous species in the Red Sea.

Cellulose Nanofibers from Softwood, Hardwood, and Tunicate: Preparation-Structure-Film Performance Interrelation.

This work reveals the structural variations of cellulose nanofibers (CNF) prepared from different cellulose sources, including softwood (Picea abies), hardwood (Eucalyptus grandis × E. urophylla), and tunicate (Ciona intestinalis), using different preparation processes and their correlations to the formation and performance of the films prepared from the CNF. Here, the CNF are prepared from wood chemical pulps and tunicate isolated cellulose by an identical homogenization treatment subsequent to either an enzymatic hydrolysis or a 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO)-mediated oxidation. They show a large structural diversity in terms of chemical, morphological, and crystalline structure. Among others, the tunicate CNF consist of purer cellulose and have a degree of polymerization higher than that of wood CNF. Introduction of surface charges via the TEMPO-mediated oxidation is found to have significant impacts on the structure, morphology, optical, mechanical, thermal, and hydrophobic properties of the prepared films. For example, the film density is closely related to the charge density of the used CNF, and the tensile stress of the films is correlated to the crystallinity index of the CNF. In turn, the CNF structure is determined by the cellulose sources and the preparation processes. This study provides useful information and knowledge for understanding the importance of the raw material for the quality of CNF for various types of applications.